Anxiolytic effects of Enterococcus faecalis 2001 on a mouse model of colitis

Ulcerative colitis (UC) is a refractory inflammatory bowel disease, which is known to cause psychiatric disorders such as anxiety and depression at a high rate in addition to peripheral inflammatory symptoms. However, the pathogenesis of these psychiatric disorders remains mostly unknown. While prior research revealed that the Enterococcus faecalis 2001 (EF-2001) suppressed UC-like symptoms and accompanying depressive-like behaviors, observed in a UC model using dextran sulfate sodium (DSS), whether it has an anxiolytic effect remains unclear. Therefore, we examined whether EF-2001 attenuates DSS-induced anxiety-like behaviors. Treatment with 2% DSS for seven days induced UC-like symptoms and anxiety-like behavior through the hole-board test, increased serum lipopolysaccharide (LPS) and corticosterone concentration, and p-glucocorticoid receptor (GR) in the prefrontal cortex (PFC), and decreased N-methyl-d-aspartate receptor subunit (NR) 2A and NR2B expression levels in the PFC. Interestingly, these changes were reversed by EF-2001 administration. Further, EF-2001 administration enhanced CAMKII/CREB/BDNF-Drebrin pathways in the PFC of DSS-treated mice, and labeling of p-GR, p-CAMKII, and p-CREB showed colocalization with neurons. EF-2001 attenuated anxiety-like behavior by reducing serum LPS and corticosterone levels linked to the improvement of UC symptoms and by facilitating the CAMKII/CREB/BDNF-Drebrin pathways in the PFC. Our findings suggest a close relationship between UC and anxiety.


Materials and methods
All experiments were approved by the Ethics Committee of Animal Experiments at the International University of Health and Welfare (Ohtawara, Japan; approval number: 19015, 22009).All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the guidelines established by the Animal Research Committee of the International University of Health and Welfare and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.Efforts were made to minimize the suffering of the animals and reduce the number of animals used in the experiments.Measurements of all experiments and analyses were performed in a blinded manner.

Elevated plus-maze test
To investigate changes in anxiety-like behavior, mice were tested using the elevated plus-maze paradigm (EPM-04M; Muromachi Kikai).The apparatus was elevated 40 cm from the ground and the maze consisted of two opposing open arms (30 cm × 6 cm × 0.3 cm) and two opposing enclosed arms (30 cm × 6 cm × 15 cm) that were connected by a central platform (6 cm × 6 cm, 70 lx), thus forming the shape of a plus sign.Each mouse was placed on a left front corner, and the distance that the mouse moved in the apparatus was recorded for 5 min by an overhead color CCD camera that tracked the center of the mouse.Moreover, the number of entries into and the time spent in open or enclosed arms were also recorded.Data from the CCD camera were collected through a custom designed interface (CAT-10; Muromachi Kikai) as a reflection signal.All of the data were analyzed and stored in a personal computer using analytical software (Comp ACT HBS; Muromachi Kikai).The results were calculated as mean ratios of the time spent or distance in the open arms to the total time spent or distance in both the open and enclosed arms.Moreover, the total number of entries into the open arms was also measured.

Western blotting
Western blotting was performed according to the experimental protocol shown in Figs.1a and 5a.Brain samples were collected from mice on day seven of DSS treatment.Moreover, mice were treated with water or EF-2001 for 20 days and sacrificed by decapitation 24 h after the last administration.The brain was immediately removed and the PFC, hypothalamus, amygdala, or ventral hippocampus were dissected quickly by a mouse brain slicer (Muromachi Kikai, Tokyo, Japan) to produce coronal sections of 1 mm thickness.We used samples from mice that had not undergone any behavioral tests.The brain atlas of Paxinos and Franklin was used as a reference to guide all dissections 51 .Protein isolation and western blotting were performed as described in prior works [52][53][54] .After electrophoresis, proteins were transferred electrically from the gel onto a polyvinylidene difluoride membrane using a semi-dry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA).The blots were blocked for 30 min with 5% skim-milk in Tris-buffered saline supplemented with 0.01% Tween-20 (TBST).Subsequently, membranes were cut between 50 and 75 kDa, and probed with antibodies except for p-CAMKII, t-CAMKII, and BDNF against phosphorylated (p)-GR (Ser211) (1:1000; Cell Signaling Technology, Danvers, MA, USA, #4161), total (t)-GR (1:200; Santa Cruz Biotechnology, Dallas, TX, USA, sc-393232), NR1

Measurement of serum LPS and corticosterone concentration
ELISA was performed according to the experimental protocol shown in Fig. 5a.Mice were treated with water or EF-2001 for 20 days and decapitated 24 h after the last treatment.The blood samples were collected from the decapitated mice into spitz tubes for serum isolation (Eiken Chemical Co., Ltd., Tokyo, Japan, #HC1100), centrifuged at 4 °C, 15 min, 2380 × g, and the supernatant was collected as a serum sample and stored at − 80 °C until the day of measurement.LPS and corticosterone concentrations were quantified using the Mouse Anti-LPS IgG Antibody Assay Kit (Chondrex, USA, WA, #6106) and Corticosterone ELISA Kit AssayMax (AssayPro, USA, MO, #EC3001-1), respectively.

Immunohistochemical study
Immunohistochemical study was performed according to the experimental protocol shown in Fig. 5a.Mice were sacrificed 24 h after the last administration of EF-2001.Brain samples were collected as described in prior works 41,42,55,56 .The brains were cut into 50-μm sections from the bregma − 1.40 mm to − 2.00 mm using a cryostat (Leica CM3050, Leica Biosystems, Tokyo, Japan).

Statistical analysis
The results of the experiments are expressed as mean ± standard error of the mean (SEM).The statistical significance of differences was determined using Student's t-test for two-group comparisons.The significance of differences was determined via one-or two-way analysis of variance (ANOVA), followed by the Tukey-Kramer test or Bonferroni test for multiple-group comparisons.The statistical significance of differences in DAI scores was assessed using a non-parametric Mann-Whitney test for two-group comparisons or a non-parametric Kruskal-Wallis test followed by the Dunn test for multiple-group comparisons.The criterion of significance was set at p < 0.05.When the main effect of group or time was significant without interaction, we performed an exploratory and limited pairwise post-hoc comparison, consistent with our a priori hypothesis.All statistical analyses using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) were performed by investigators other than the experimenters to avoid bias and ensure blinding.
Mice treated with 2.5% DSS were observed to move less, based on the distance covered, than controls (Supplementary Fig. S1).To exclude the possibility of decreased locomotor activity associated with exacerbated IBD-like symptoms, the concentration of 2% DSS was used in subsequent experiments in this study of anxiety associated with IBD.

Time-dependent effects of DSS on DAI scores, colon length, and anxiety-related behaviors in mice
As shown in Fig. 2, diarrhea, bloody stool, and shortened colon length were observed on days five and seven of 2% DSS treatment, but not on day three

Effects of DZP or TDS on anxiety-related behaviors in 2% DSS-treated mice
The effects of DZP and TDS-clinically used anxiolytic drugs-on anxiety-like behaviors in DSS-treated mice were examined.The results showed that acute administration of DZP (1 mg/kg) led to significant improvements in the outcomes assessed, effectively addressing reductions in the number or duration of head-dips, the time spent in the central area, the number of open-arm entries, and the percentage of distance traveled or time spent in the open arm in the 2% DSS-treated group in the hole-board and elevated plus-maze tests [one-way ANOVA: F (4, 67) = 17.52, p < 0.0001, Fig. 3a; F (4, 67) = 10.33,p < 0.0001, Fig. 3b; F (4, 67) = 5.942, p = 0.0004, Fig. 3c; F (4, 67) = 16.63,p < 0.0001, Fig. 3e; F (4, 67) = 14.12, p < 0.0001, Fig. 3f; F (4, 67) = 7.654, p < 0.0001, Fig. 3g].In contrast, acute administration of TDS (1, 3 mg/kg) had a significant restorative effect only with regard to the decreased time spent in the central area, with no effect on other changes in the hole-board and elevated plusmaze tests (Fig. 3c,d).

Changes in phosphorylation levels of GR in the brain of 2% DSS-treated mice
We examined the changes in phosphorylation levels of GR in the brain regions that are closely related to the development of anxiety, such as the PFC, amygdala, hypothalamus, and ventral hippocampus.According to the results, 2% DSS-treated mice showed an increase in phosphorylation levels of GR in the PFC and ventral hippocampus, but not in the amygdala and hypothalamus, compared to the water group (Student's t-test: t = 3.125, df = 10, p = 0.0108, Fig. 4a; t

Effects of EF-2001 on serum LPS and corticosterone concentration and p-GR in the brain
Serum corticosterone levels in the 2% DSS-treated mice were significantly elevated compared to those in the water group (serum LPS concentration in the 2% DSS-treated group was slightly higher (p = 0.1096) compared to that in the water group), while these changes were prevented by EF  6d].Additionally, we observed that the p-GR (Ser211) labeling was colocalized in the neurons, but not in the astrocytes and microglia, of the PFC of DSS-treated mice (Fig. 6e).

Effects of EF-2001 on NMDAR signaling and synaptic plasticity-related proteins
As shown in Fig. 7, the expression levels of NR2A and NR2B in the PFC of 2% DSS-treated mice were significantly lower compared to those in the water group (the expression levels of p-CAMKII and BDNF in the PFC of the 2% DSS-treated group decreased slightly (p-CAMKII: p = 0.0755; BDNF: p = 0.052) compared to those in the water group), whereas these changes were reversed by EF-2001   7l].Additionally, we observed that the p-CAMKII and p-CREB labeling were colocalized in the neurons of the PFC (Fig. 7m,n).

Discussion
A high prevalence of psychiatric disorders such as depression and anxiety has been reported among patients with IBD, but the mechanisms underlying the relationship between intestinal inflammation and anxiety symptoms remain unclear.In this study, we examined the effects of EF-2001 on IBD-like physiological changes  As mentioned above, a high rate of neuropsychiatric disorders has been reported to occur in patients with IBD 1-5 , but the underlying mechanisms remain unclear; other studies have reported that DSS-treated mice, commonly used in modeling UC, show UC findings, such as diarrhea and bloody stools, and exhibit anxietylike behaviors 28,29 .In the present study, 2% DSS-treated mice exhibited UC-like symptoms such as diarrhea, hematochezia, and shortened colon length as well as anxiety-like behaviors in the hole-board and elevated plusmaze tests (Figs. 1, 2), without affecting locomotor activity (Supplementary Fig. S1).The effects of the clinically used anxiolytics DZP and TDS on anxiety-like behaviors in DSS-treated mice were also examined.The results showed that acute administration of DZP suppressed anxiety-like behaviors observed in the 2% DSS-treated group in both the hole-board and elevated plus-maze tests, while acute administration of TDS suppressed only the decrease in behavioral time in the central area in the hole-board test in the 2% DSS-treated group, but had no effect on other anxiety-like behaviors (Fig. 3).In clinical practice, TDS is less effective as a single dose than DZP and more effective when administered chronically; the results of this study appear to reflect this clinical fact 57 .Activation of GR in the PFC, hypothalamus, amygdala, and ventral hippocampus is known to induce anxiety-like behaviors via diverse mechanisms [12][13][14][15] .DSS-treated mice showed increased phosphorylation levels of GR in the PFC and ventral hippocampus (Fig. 4).From these results, we suggest that DSS-treated mice are a useful model of anxiety accompanying UC, as demonstrated the three validities (surface, predictive, and construct validities) which must be fulfilled when building a model animal 58,59 .Moreover, it has been suggested that increased phosphorylation of GR in the PFC and ventral hippocampus may be implicated in anxiety-like behaviors.
Previous studies demonstrated that EF-2001 administration prevented DSS-induced depression-like behavior and UC-like symptoms, with inflammation suppressed in the hippocampus and rectum 32 .In the present study, EF-2001 prevented DSS-induced UC-like symptoms and anxiety-like behaviors in the hole-board test, while it did not suppress anxiety-like behaviors in the elevated plus-maze test (Fig. 5).Moreover, administration of EF-2001 showed no changes in normal mice in both the hole-board and elevated plus-maze tests (Supplementary Fig. S5).As the elevated plus-maze test is thought to assess anxiety-like behavior under strong fear conditions compared to the hole-board test 60,61 , it may be that EF-2001 administration does not mitigate anxiety-like behavior associated with fear, but only that caused by mild stress.
In patients with UC, the intestinal microbiota is thought to be imbalanced, with a relative increase in gramnegative bacteria and a decrease in tight junctions between cells in the intestinal tract, leading to increased migration of inflammatory cytokines and LPS secreted by gram-negative bacteria into the blood 7,8 .In animal studies, intraperitoneal administration of LPS has been reported to increase blood corticosterone [9][10][11] .In the present study, DSS-treated mice were found to have increased serum LPS levels and a significant increase in serum corticosterone levels, and these changes were significantly prevented by EF-2001 administration (Fig. 6a,b).These results suggest that UC-like symptoms caused LPS to migrate from the intestinal tract into the blood and elevated the blood corticosterone levels.Moreover, EF-2001 administration prevented an increase in p-GR levels in the PFC of DSS-treated mice, but not in the ventral hippocampus (Fig. 6c,d).We found that phosphorylation of GR was localized to neurons in the PFC, but not to astrocytes and microglia (Fig. 6e).The ventral hippocampus region is known to be related to anxiety under conditions involving fearful stimuli, which can lead to high stress 62,63 .In the present study, EF-2001 showed no anxiolytic effect in the elevated plus-maze test, which assesses anxiety under fearful conditions.This result may be related to the fact that EF-2001 did not suppress the increase in phosphorylation levels of GR in the ventral hippocampus.From these findings, we suggest that the anxiolytic effect of EF-2001 may be associated with the decreased phosphorylation level of GR in the PFC neurons via the prevention of the increase in serum LPS and corticosterone levels.www.nature.com/scientificreports/Activation of GR by long-term stress exposure has been reported to cause abnormal synaptic plasticity 16 .Increased corticosterone causes decreased expression levels of NR2A, NR2B, BDNF, and synaptic plasticity-related proteins [17][18][19] , and these changes in the PFC are associated with the development of anxiety-like behavior [20][21][22] .Activated NMDAR phosphorylates CAMKII, which in turn phosphorylates CREB, resulting in BDNF and drebrin translation through CREB activation [23][24][25] .Activation of BDNF signaling by the tropomyosin receptor kinase B agonist 7,8-dihydroxyflavone produces anxiolytic effects 26 .It has been reported that increased expression of drebrin in the PFC plays an important role in anxiolytic effects 27 .BDNF and Drebrin expression in the PFC has been suggested to correlate with anxiety-like behaviors in rodents 27,64,65 .In the present study, the expression levels of p-CAMKII and BDNF in the PFC of DSS-treated mice showed a decreasing tendency, and the expression levels of NR2A and NR2B significantly decreased, while these changes were reversed by EF-2001 treatment (Fig. 7b-d,f).Furthermore, EF-2001 treatment increased the expression levels of p-CREB and drebrin in the PFC of DSS-treated mice (Fig. 7e,j).In contrast, the expression levels of NR1, synaptophysin, SNAP25, PSD95, MAP2, and NeuN in the PFC were not significantly altered.We found that p-CAMKII and p-CREB were localized to neurons in the PFC (Fig. 7m,n).From these findings, we suggest that the anxiolytic effects of EF-2001 may be associated with activation of the CAMKII/CREB/BDNF-Drebrin pathways in the PFC neurons via normalized expression levels of NR2A and NR2B.
Despite these novel results, this study had some limitations that must be noted.In the present study, we focused on the relationship between anxiety behavior and activation of GR in the brain.However, we consider that studies focusing on these relationships alone are insufficient to elucidate the pathogenesis of anxiety accompanying UC.The reason is that there have been many studies focusing on neural activity in the brain and anxiety behavior [66][67][68][69] , which we did not examine in this study.Moreover, it is unclear as to how elevated serum LPS contributes to elevated corticosterone and how EF-2001 acts on changes in serum LPS.Furthermore, it has been reported that changes in the intestinal microbiota affect the brain via the vagus nerve, resulting in anxiety-like behavior 70 .To determine whether the microbiota-gut-brain axis via the vagus nerve contributes to the anxiolytic

Conclusion
As summarized in Fig. 8, the results from our DSS-induced UC model with mice show that elevated serum LPS and corticosterone levels activate GR in the PFC, resulting in anxiety-like behaviors.EF-2001 inhibits these changes by preventing enteritis symptoms, and produces anxiolytic effects via the activation of synaptic plasticity in the PFC.These results suggest a close relationship between IBD and anxiety, and provide important evidence for a mechanism by which the attenuation of intestinal inflammatory symptoms in UC may reduce the risk of psychiatric disorders.

Figure 1 .
Figure 1.Effect of DSS concentration on ulcerative colitis-like findings and anxiety-like behaviors.(a) Experimental time course for assessment of inflammation, behavioral tests, western blotting, ELISA, and immunohistochemical of experimental protocols.Changes in rectal bleeding (b), stool consistency (c), colon length (d), head-dip counts (e), head-dip duration (f), total time spent in the central area (g), number of openarm entries (h), percentage of distance traveled (i) or time spent (j) in the open arm in dextran sulfate sodium (DSS)-treated mice on day seven.Bars represent means ± standard error of mean (SEM).*p < 0.05 and **p < 0.01 vs. water group (n = 14 per group).
All experiments were approved by the Ethics Committee of Animal Experiments at the International University of Health and Welfare (Ohtawara, Japan; approval number: 19015, 22009).All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the guidelines established by the Animal Research Committee of the International University of Health and Welfare and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.Efforts were made to minimize the suffering of the animals and reduce the number of animals used in the experiments.

Figure 2 .
Figure 2. Time-course of DSS treatment for ulcerative colitis-like findings and anxiety-like behaviors.Timecourse of rectal bleeding (a), stool consistency (b), colon length (c), head-dip counts (d), head-dip duration (e), total time spent in the central area (f), number of open-arm entries (g), percentage of distance traveled (h) or time spent (i) in the open arm in dextran sulfate sodium (DSS; 1.5%)-treated mice on days three, five, and seven.Bars represent means ± standard error of mean (SEM).*p < 0.05 and **p < 0.01 vs. water group (n = 20 per group).

Figure 3 .
Figure 3. Effects of anxiolytics on DSS-induced anxiety-like behaviors.Effect of acute treatment with diazepam (DZP) or tandospirone (TDS) on head-dip counts (a), head-dip duration (b), total time spent in the central area (c), number of open-arm entries (e), percentage of distance traveled (f) or time spent (g) in the open arm in dextran sulfate sodium (DSS)-treated mice.(d) and (h) Representative activity traces in the hole-board test (d) and the elevated plus-maze test (h).The central area in the hole-board test is indicated by a green rectangle.Bars represent means ± standard error of mean (SEM).**p < 0.01 vs. water-treated water group, # p < 0.05 and ## p < 0.01 vs. water-treated DSS group (n = 13-16 per group).

Figure 4 .
Figure 4. Changes in phosphorylation levels of glucocorticoid receptors in anxiety-related brain regions.Changes in phosphorylation and expression levels of glucocorticoid receptor (GR) in the prefrontal cortex (PFC) (a,b), amygdala (c,d), hypothalamus (e,f), and ventral hippocampus (g,h) of DSS-treated mice.Quantification of the normalized values of p-GR and t-GR with t-GR and β-actin, respectively.Original immunoblot images were shown in Supplementary Fig. S2.Bars represent means ± standard error of mean (SEM).*p < 0.05 and **p < 0.01 vs. water group (n = 6 per group).

Figure 5 .
Figure 5. Effects of EF-2001 on DSS-induced ulcerative colitis-like findings and anxiety-like behaviors.(a) Experimental time course for assessment of inflammation, behavioral tests, western blotting, ELISA, and immunohistochemical of experimental protocols.Effects of chronic treatment with Enterococcus faecalis 2001 (EF-2001) on rectal bleeding (b), stool consistency (c), colon length (d), head-dip counts (f), head-dip duration (g), total time spent in the central area (h), the number of open-arm entries (j), percentage of distance traveled (k) or time spent (l) in the open arm in dextran sulfate sodium (DSS)-treated mice.(e) Representative colon images from each group are shown.(i) and (m) Representative activity traces in the hole-board test (i) and the elevated plus-maze test (m).The central area in the hole-board test is indicated by a green rectangle.Bars represent means ± standard error of mean (SEM).**p < 0.01 vs. water-treated water group, # p < 0.05 and ## p < 0.01 vs. water-treated DSS group (n = 15 per group).

Figure 6 .
Figure 6.Effects of EF-2001 on serum LPS and corticosterone, and p-GR in the brain.Effects of chronic treatment with Enterococcus faecalis 2001 (EF-2001) on increased serum LPS (a) and corticosterone (b) concentration, and enhanced p-GR in the prefrontal cortex (PFC) (c) and ventral hippocampus (d) of DSStreated mice.Quantification of the normalized values of p-GR with t-GR.Original immunoblot images were shown in Supplementary Fig. S3.(e) Microscopy images of p-GR (Ser211) (green), DAPI (blue), and NeuN, GFAP or Iba1 (red) immunostaining in the PFC of DSS-treated mice.Bars represent means ± standard error of mean (SEM).*p < 0.05 and **p < 0.01 vs. water-treated water group, # p < 0.05 and ## p < 0.01 vs. water-treated DSS group (n = 6-8 per group).